Expression of two consecutive genes of a secondary metabolic pathway in transgenic tobacco: molecular diversity influences levels of expression and product accumulation

We have created a population of transgenic tobacco plants carrying cDNAs encoding two consecutive enzymes from early stages in monoterpenoid alkaloid biosynthesis in Catharanthus roseus. The cDNAs, encoding tryptophan decarboxylase ((tdc)) and strictosidine synthase (str1) together with a selectable marker gene, were introduced on a single transforming plasmid into tobacco leaves by particle bombardment. Analysis of 150 independent transgenic plants at the DNA and RNA levels demonstrated a range of integration events and steady-state transcript levels for the tdc and str1 transgenes. Southern blot analysis indicated that the tdc and str1 transgenes were integrated at least once in all 150 transformants giving a 100% co-integration frequency of the two unselected genes carried on the same plasmid. A comparison of Southern and northern data suggested that in 26% of the plants, both tdc and str1 transgenes were silenced, 41% demonstrated a preferential silencing of either the tdc or the str1 transgene, with the remaining 33% of the plants expressing both transgenes. We observed no clear correlation between the number of integration events of a specific transgene and the levels of accumulated transcript. Twenty plants representing the range of molecular diversity in the transgenic population were selected for further analysis. Seeds were collected from self-fertilised transformants and germinated on medium containing kanamycin. Seedlings were harvested after 7 weeks and TDC and STR1 enzymatic assays were carried out. We observed a 24- and 110-fold variation in levels of TDC and STR1 activities, respectively. Our data correlate molecular diversity with biochemistry and accumulation of end-product and provide a detailed molecular and biochemical characterization of transgenic plants transformed with a single plasmid carrying two genes of secondary metabolism.